Intermediates for the production of cephalosporin compounds

ABSTRACT

CEPHALOSPORINS HAVING A 7-A-AMINOARYLACETAMIDO SUBTITUENT AND A NUCLEOPHILE ON THE 3-METHYL GROUP ARE PREPARED BY REACTION OF THE NUCLEOPHILE WITH THE NEW INTERMEDIATES, 7-(D-2,2-DIMETHYL-3-NITROSO-5-OXO4-ARYL-1-IMIDAZOLIDINYL-3-BROMOMETHYL-3 -CEPHEM4-CARBOXYLIC ACID SULFOXIDES AND THEIR EASILY HYDROLYZED ESTERS SUCH AS TRIMETHYLSILYL AND 2,2,2-TRICHLOROETHYL, FOLLOWED BY REMOVAL OF THE BLOCKING GROUPS, AS BY HYDROGEN CHLORIDE IN DIOXANE OR GLACIAL ACETIC ACID. THE INTERMEDIATES THEMSELVES ARE PREPARED BY BROMINATION, AS WITH N-BROMOSUCCINIMIDE, OF THE CORRESPONDING 3-METHYL COMPOUNDS.

United States Patent 3,767,655 INTERMEDIATES FOR THE PRODUCTION OF CEPHALOSPORIN COMPOUNDS Lee C. Cheney and William J. Gottstein, Fayetteville, N.Y., assignors to Bristol-Myers Company, New York,

No brawing. Filed Sept. 30, 1971, Ser. No. 185,384 Int. Cl. C07d 99/24 U.S. Cl. 260--243 C 11 Claims ABSTRACT OF THE DISCLOSURE Cephalosporins having a 7-a-aminoarylacetamido substituent and a nucleophile on the B-methyl group are prepared by reaction of the nucleophile with the new intermediates, 7 (D 2,2 dimethyl 3 nitroso 5 oxo- 4-aryl 1 imidazolidinyl 3 bromomethyl 3 cephem- 4-carboxylic acid sulfoxides and their easily hydrolyzed esters such as trimethylsilyl and 2,2,2-trichloroethyl, followed by removal of the blocking groups, as by hydrogen chloride in dioxane or glacial acetic acid. The intermediates themselves are prepared by bromination, as with N-bromosuccinimide, of the corresponding 3-methyl compounds.

BACKGROUND OF THE INVENTION Field of the invention This invention includes new processes and new intermediates for the production of compounds of the cephalosporin class and particularly cephalosporins having a nucleophilic substituent on the methylene group at the 3-position.

Description of the prior art Applicants starting materials are the 3-methyl cephalosporins of the cephalexin type having a particular blocking group on the a-amino group as described in their, prior, copending applications Ser. No. 27,441, filed Apr. 10, 1970 and now abandoned, and Ser. No. 78,165 filed Oct. 5, 1970 and issued Jan. 30, 1973 as U.S. 3,714,146; for a brief summary of previously used blocking groups see page of Great Britain 1,241,656.

U.S. Pat. 3,507,861 describes such 3-methyl cephalosporins, including the well-known cephalexin, which is also described, for example, in J. Med. Chem., 12, 310- 313 (1969), in Great Britain 1,174,335, in South Africa 67/1260 (Farmdoc 28654), in Japan 16871/66 (Farmdoc 23231), in Belgium 696,026 (Farmdoc 29494).

The conversion of a penicillin sulfoxide ester (by heating in the presence of a strong acid) to the corresponding ester of a similarly N-acylated derivative of 7-ADCA has been reported in U.S. Pat. 3,275,626 and in J. Amer. Chem. Soc., 85, 1896 (1963) and 91(6), 1401-1407 (1969). Variations of this process are given in Netherlands 68/06532 (Farmdoc 34685) and Netherlands 68/ 06533 (Farmdoc 34686). In those patents the side-chain is usually that of a fermentable penicillin such as penicillin G or V (although see Column 7 of U.S. Pat. 3,275,- 626) and the product is an ester which must be cleaved, as by hydrogenation, to produce the active, free acid form of the final derivative of 7-ADCA. Great Britain 1,174; 335 in Example 3 describes the application of this sulfoxide rearrangement to an ester of ampicillin sulfoxide in which the u-amino group is also blocked, that is, 6- [N-(2,2,2 trichloroethylcarbonyl D a amino ozphenylacetamido]penicillanic acid sulfoxide 2,2,2 trichloroethyl ester, by heating and then use of zinc and acetic acid to remove the two blocking groups and thus produce cephalexin.

3,767,655 Patented Oct. 23, 1973 The art contains numerous additional descriptions of penicillin sulfoxides and their preparation, as described for example, by Chow et al., J. Org. Chem. 27, 1381 (1962), by Guddal et al., Tetrahedron Letters No. 9, 381 (1962), by Essery et al., J. Org. Chem., 30, 4388 (1965) who included ampicillin sulfoxide, and in U.S. Pats. 3,197,466 and 3,544,581.

The reaction product of acetone with cephaloglycin, but not with cephalexin, is described in U.S. Pat. 3,303,- 193. The reaction with acetone of certain ring-substituted cephalexins is described in general terms in U.S. Pats. 3,489,750, 3,489,751 and 3,489,752. 7-a-aminothienylacetamidocephalosporanic acids are described in U.S. Pats. 3,560,489 and 3,352,858 and are converted to corresponding compounds having a 3-methyl group (cephalexin type) by the usual hydrogenation as first described in this field, for example, in U.S. Pat. 3,124,576 and see Great Britain 1,174,335 (and see also Canada 830,995).

The nucleophiles used to replace the bromine atom of the 3-bromomethyl substituent in the process of the present invention include acetate and the thiols described as displacing acetoxy in U.S. Pat. 3,516,997 and the nucleophiles defined and described in Great Britain 1,241,657.

The preparation of various 7-[a-amino-arylacetamido]- cephalosporanic acids and the corresponding desacetoxy compounds in which aryl represents unsubstituted or substituted phenyl or 2- or 3-thienyl is described, for example, in British specifications 985,747, 1,017,624, 1,054,- 806 and 1,123,333, in Belgian Pat. 696,026 (Farmdoc No. 29,494), in U.S. Pats. 3,311,621, 3,352,858, 3,489,- 750, 3,489,751, 3,489,752 and 3,518,260, in Japanese Pat. 16871/66 (Farmdoc 23,231), by Spencer et al., J. Med. Chem., 9 (5), 746-750 (1966) and by Kurita et al., J. Antibiotics (Tokyo) (A) 19, 243-249 (1966) and I see also US. Pat. 3,485,819.

Netherlands Pats. 68/ 11676 (Farmdoc 36,349) and 68/ 12382 (Farmdoc 36,496) and U.S. Pats. 3,489,750 and 3,489,751 disclose ring-substituted cephaloglycins.

Various 7 [a amino arylacetamido]cephalosporins in which one hydrogen of the a-amino group is replaced by a carbonyl group which is attached in turn to another moiety have been reported. The earliest were the cephaloglycin and cephalexin precursors in which use was made of a common peptide blocking group such as carbobenzyloxy as illustrated by U.S. Pat. 3,364,212, Belgian Pat. 675,298 (Farmdoc 22,206), South African Pat. 67/ 1260 (Farmdoc 28,654) and Belgian Pat. 696,026 (:Farmdoc 29,494). Related compounds include those of U.S. Pats. 3,303,193 and 3,311,621 and 3,518,260.

Various cephalosporins, including cephalosporin C on occasion but not cephaloglycin, have been reacted with nucleophilic, aromatic mercaptans to produce compounds having the structure S Acyl-NH-CH-CH CHz O: N CHz--SAr 0 (EOOH *In U.S. Pat. 3,278,531 Ar is phenyl or certain substituted phenyls or certain aromatic heterocyclic rings named, for example, in column 5. Similar nucleophiles, e.g. 2-mercaptopyrimidines, are disclosed in U.S. 3,261,832 and 3,502,665, all issued to Glaxo. A parallel disclosure is 3 found in Great Britain 1,109,525 to Ciba, e.g. in definition 11 for R Additional nucleophiles of this type were disclosed by Fujisawa in Belgium 714,518 (Farmdoc 35,307; Netherlands 68/06129 and South Africa 2695/ 68), in Canada 818,501 (Farmdoc 38,845), in Great Britain 1,187,323 (Farmdoc 31,936; Netherlands 67/ 14888) and especially in US. 3,516,997 (Farmdoc 34,328; Netherlands 68/05179) which includes the compound named cefazolin, which has a tetrazolylacetyl sidechain on the 7-amino group and a S-methyl-thiadiazolylthiomethyl group at the 3-position and is described at some length in the scientific literature, eg in Antimicrobial Agents and Chemotherapy-4969, American Society for Microbiology, Bethesda, Md. at pages 236-243 and in J. Antibiotics (Japan) 23(3), 131-148 (1970). Various cephalosporins having the structure in which acyl represents various sidechains including aminophenylacetyl have been described in some of the above and by Glaxo in Belgium 734,532 (Farmdoc 41,619) and in Belgium 734,533 (Farmdoc 41,620).

Cephalosporins having the structure s Acyl-NH-CH-(EH (13H? O=CN CCH2X i coon where X includes are disclosed in some of the above and in US. 3,239,515, 3,239,516, 3,243,435, 3,258,461, 3,431,259 and 3,446,803.

Related publications in the scientific literature inciude J. Med. Chem. 8, 174-181 (1965) and J. Chem. Soc. (London) 1595-1605 (1965), 5015-5031 (1965) and 1959-1963 (1967).

One of the most extensive series of chemical publications in the cephalosporin field has come from scientists of Eli Lilly and Company; each paper gives a citation of the previous paper. The latest in series appears to be XXI appearing in J. Org. Chem. 36(9), 1259-1267 (1971).

Various procedures for reducing cephalosporin sulfoxides and converting esters to free acids are disclosed in South Africa 69/4766 (Eli Lilly).

The preparation of 2-(2-methyl-3-butenyl)-3-bromomethyl-7-phenoxyacetamido-A. cephem-4-carboxylate is described in Example 3 of South Africa 69/8494 (Eli Lilly) and it is reacted with methyl mercaptan in Example 4 and that product is then isomerized to the A compounds (3-methylmercapto) in Example 5.

The preparation from 3-hydroxymethyl cephalosporins of various 3-halomethyl cephalosporins, e.g. diphenylmethyl 3-bromomethyl-7fi-2-thienylacetamido-ceph-3-em- 4-carboxylate, is described in Great Britain 1,241,658 (Glaxo) and Great Britain 1,241,657 describes their reaction with nucleophiles. For the 7-substituent, meaning the carboxylic acid residue attached to the 7-amino group, Great Britain 1,241,658 describes in general terms a vast number of substituents including the following statement as to the eleventh and last such class:

(Xi) a-Substituted carboxylic acid acyl groups, where the oc-substituent is an amino, substituted amino [c.g.

acylamido or a group obtained by reacting the amino group and/or acylamido group(s) with an aldehyde or ketone e.g. acetone or methyl ethyl ketone], hydroxy, carboxy, esterified carboxy, cyano, halogeno, acyloxy (e.g. formyloxy or lower alkanoyloxy) or etherified hydroxy group. The carboxylic acid may be aliphatic, e.g. an a.- substituted paraffinic acid, or araliphatic, e.g., an a-substituted phenylacetic acid. Acyl groups of this character include particularly the group ArCH(X)CO where Ar is a phenyl or substituted phenyl group and X is an aminoor substituted amino group. See also Great Britain 1,241,655 and 1,241,656.

Glaxos South Africa 70/5851 (claiming priority from a series of applications of which the earliest is G.B. 42502/ 69 filed Aug. 26, 1969) is concerned with brominetion of cephalosporins and subsequent reactions. Thus, a representative example discloses the preparation and bromination of 2,2,2-trichloroethyl 3-methyl-7-(2,2,2- trichloroethoxycarbonylamino)ceph-3-em-4 carboxylate sulfoxide to give 2,2,2-trichloroethyl 3-bromomethyl-7- (2,2,2-trichloroethoxycarbonylamino)ceph-3-ern 4 carboxylate sulfoxide which was subsequently reacted with methanethiol to produce the fully blocked 3-methylthiomethyl compound which was converted by treatment with zinc dust in formic acid to 7-(D-2-amino-2-phenylacetamido -3-methyl-thiomethyl-ceph-3-em-4-carboxylic acid and reaction with sodium acetate.

Bromination is also described schematically in US. Pat. 3,544,581.

US. Pat. 3,637,678 issued Jan. 25, 1972 discloses 3- bromomethyl-n -cephalosporin ester intermediates, and A -3-functionalized cephalosporin esters prepared by (a) brominating the 3-methyl group of a A -desacetoxycephalosporin ester, and then (b) reacting the resulting 3-brornomethyl-A -cephalosporin ester with a nucleophilic reagent to displace the bromine with the nucleophile and to form 3-(functionalized-methyl)-A -cephalosporin esters which are useful as intermediates in the production of cephalosporin antibiotics, e.g., from penicillin starting materials.

Certain 3-bromomethy1 cephalosporins are disclosed in US. 3,647,788 issued Mar. 7, 1972, in US. 3,668,203 issued June 6, 1972 in US. 3,705,897 issued Dec. 12, 1972, in US. 3,708,480 issued Jan. 2, 1973, in Lillys Belgium 748,055 (Farmdoc 71,847R) and in J. Med. Chem. 14(11), 1136-1138 (1971) and the earlier publications cited therein with reference to their compound 1.

SUMMARY OF THE INVENTION The present invention includes, but is not limited to, the novel intermediates (I, II and III below) and the separate processes (A through D below) which may be summarized 0 R R=H or an easily hydrolyzed gse such as 011C 0113- or as illustrated by NBS (CHCb) hv A brominating agent l 13. A nucleophlle as illustrated by t t KSC\ /JJCH3 ll 0 i t ON-N N NN CH S y? P) CH on. on. 3

C. A sulfoxide reducing agent as illustrated by NB2S2O4 CH COOl Ar\ /H O t' t S ON-N N- tt CH3 CH3 O N\ CH; s & on:

L O bOzR D. An unblocklng agent lll as illustrated by H01 in dioxane or CHsCOOH l S Ar-o11o NH- l l N-N 0 N on s ('5 on :u 1" T I OOH The bromination of Process A above may be effected by any convenient system capable of generating bromine atoms such as bromine itself, or a bromine transfer agent e.g. an N-bromoamido or an N-bromoimide. The N-amide or N-bromoimide may include a cyclic system, the amide or imide linkage forming part of the cyclic system; examples of such N-bromoamides include caprolactam and examples of such N-bromoimides include the 1,3-dibromo- 5,5-diloweralky1 hydantoins e.g. 1,3dibromo-5,5-dimethylhydantoin; 1,3-dibromo-5-ethyl-S-methylhydantoin; 1,3- dibromo-S-isopropyl-S-methylhydantoin, N-bromosuccinimide, N-bromophthalimide etc. Other useful N-bromoamides include N-bromo lower alkanoamides e.g. N-bromoacetamide. Another useful brominating agent is 1,3,5- tribromo-1,2,4-triazole. By reason of their availability, particularly preferred brominating agents include N-bromosuccinimide and 5,5-dimethyl-l,S-dibromohydantoin.

The various brominating agents require initiation in order to generate bromine atoms and suitable initiating systems include free-radical initiators such as azo compounds e.g. azobisisobutyronitrile, peroxides e.g. benzoyl peroxide, irradiation by ultra violet or visible light sources e.g. mercury arcs or tungsten lamps, or by 'y-rays emitted by C0 sources.

The brominating agent may be added as such or in suspension or solution in a suitable solvent i.e. a solvent which solubilizes the starting material and which is substantially inert under the conditions or the reaction e.g. a hydrocarbon such as benzene or a halogenated hydrocarbon particularly a chlorinated hydrocarbon e.g. chloroform, methylene chloride, 1,2-dichloroethane etc. The brominating agent is added to a solution or suspension of the cephalosporin compound in a suitable solvent e.g. a halogenated hydrocarbon such as methylene chloride, chloroform, 1,2-dichloroethane or chlorobenzene or a hydrocarbon such as benzene. The bromination may be effected at temperatures ranging from --80 to +150 C. e.g. from -20'to +l50 C., preferably from 40 to C. The course of the bromination may be followed by measurement of the consumption of brominating agent and by thin-layer chromatography. The course of the reaction may also be followed by monitoring the ultra violet absorption spectrum or optical rotation.

The bromination is preferably carried out using N- bromosuccinimide or a dibromohydantoin as the brominating agent initiated by ultra violet irradiation at a low temperature e.g. from -20 to +10 C.

The addition of small amounts e.g. up to 5% by volume of water or an aqueous solution or suspension of a weak base such as an alkali metal or alkaline earth metal salt of a weak acid e.g. sodium bicarbonate, sodium carbonate, sodium acetate or calcium carbonate has been found to assist the bromination reaction. In this way the times of initiation and reaction may be reduced and/or the yield of 3-bromomethyl compound may be increased. The aqueous solution or suspension of the weak base is preferably at a pH of 7 to 11.

The bromination may be effected under an inert atmosphere.

After the introduction of the desired nucleophilic group the l-sulphinyl group may be reduced by any convenient means. This may, for example, be eifected by reduction of the corresponding acyloxysulphonium or alkyloxysulphonium salt prepared in situ by reaction with e.g. acetyl chloride in the case of an acetoxy-sulphonium salt, reduction being effected by, for example, sodium dithionite or by iodide ion as in a solution of potassium iodide in a water miscible solvent e.g. acetic acid, tetrahydrofuran, dioxane, dimethylformamide or dimethylacetamide. The reaction may be effected at a temperature of 20 to +50 C.

Alternatively, reduction of the l-sulphinyl group may be effected by phosphorus trichloride or tribromide in solvents such as methylene chloride, dimethylformamide or tetrahydrofuran, preferably at a temperature of 20 to +50 C.

(a) 7-(D-2,2-dimethyl-3-nitroso 5 oxo-4-phenyl-1-imidazolidinyl) 3 (5 methyl-1,3,4-oxadiazol-2-ylthiomethyl)-3-cephem-4-carboxylic acid l-oxide To a solution of 1 g. (0.0023 mole) of 7-(D-2,2-dimethyl-3-nitroso-5-oxo 4 phenyl-1-imiclazolidinyl)-3- 7 methyl-3-cephem-4-carboxylic acid sulfoxide in 100 ml. of methylene chloride at 10 was added 230 mg. (0.0023 mole) of triethylamine and 250 mg. (0.0023 mole) of chlorotrimethylsilane. The reaction mixture was stirred at rt for 2 hours and cooled to 5 and 620 mg. (0.0035 mole) of N-bromosuccinimide with a catalytic amount of benzoyl peroxide was added. The solution was irradiated at 5 for 3 hours with a photofiood lamp and 370 mg. (0.0023 mole) potassium S-methyl-1,3,4-oxadiazole-2- mercaptide was added all at once. The mixture was stirred overnight at rt. The methylene chloride was evaporated and the residue was dissolved in 50 ml. of ethyl acetate and extracted with 50 ml. of sodium bicarbonate solution. The aqueous extract was layered with ethyl acetate and acidified to pH 2 with 1:1 phosphoric acid. The organic layer was washed with water and evaporated to a gum which was dried by azeotropic distillation with ethyl acetate. The residue was triturated with ether to give 250 mg. of a light yellow solid. A bioautogram of this solid against a B. subrzlis pH 6-seeded agar plate of a Brinkman silica gel plate in an acetone-methanol (1:1) system showed a biologically active sport corresponding exactly in R; value with that of authentic 7-(D-2,2-dimethyl-3-nitroso-5oxo-4-phenyl- 1 -imidazolidinyl -3- 5 methyl-1,3,4-oxadiazol 2 yl-thiomethyl)-3-cephem-4-carb-oxylic acid sulfoxide.

(b) 7-(D-2,2-dimethyl-3-nitroso 5 oxo-4-phenyl-l-imidazolidinyl)-3-(5-methyl 1,3,4 oxadiazol-Z-ylthiomethyl)-3-cephem-4-carboxylic acid To a solution of 100 mg. (0.002 mole) of the above 7 (D-2,2-dimethyl-3-nitroso-5-oxo-4-phenyl-l-imidazolidinyl) 3 (5 methyl-l,3,4-oxidazol2-ylthiomethyl)-3- cephem-4-carboxylic acid l-oxide in 2 ml. of acetonitrile at 5 was added 100 mg. of sodium dithionite and 0.11 ml. of acetyl chloride. The mixture was stirred in the cold for 1 hour. Five ml. of water and 5 ml. of ethyl acetate were added and the mixture was adjusted to pH 6.5. The mixture was acidified to pH 2 with 1:1 phosphoric acid and ethyl acetate layer was washed with water and evaporated to a gum. It was dried by azeotropic distillation with ethyl acetate. The residue was solidified with dry ether and dried over P to yield 20 mg. A bioautogram of this solid against a B. subtilis pH 6-seeded agar plate of a Brinkman silica gel plate in a 1:1 acetone-methanol system shows a biologically active spot corresponding exactly with that of authentic 7-(D-2,2-dimethyl 3-nitroso-5-oxo-4-phenyl-l-imidazolidinyl)-3-(5- methyl 1,3,4-oxadiazol-2-ylthiomethyl)-3-cephem-4-carboxylic acid.

(c) 7 (D-a-amino a phenylacetamido)-3-(S-methyl- 1,3,4-oxadiazol-2-ylthiomethyl) 3 cephem-4-carboxylic acid To a solution of two grams of 7-(D-2,2-dimethyl-3- nitroso-S-oxo-4-phenyl 1 imidazolidinyl)-3-(5-rnethyl- 1,3,4 oxadiazol 2 ylthiomethyl)-3-cephem-4-carbox ylic acid dissolved in 50 ml. of dioxane bubble in dry hydrogen chloride gas for 5 minutes. Stir the solution for 5 minutes and remove the solvent at 30 at mm. Stir the residue with ethyl acetate and collect about 1.9 g. of crude 7-(D-a-amino-a-phenylacetamido) 3 (S-methyl- 1,3,4 oxadiazol-Z-ylthiomethyl) 3 cephem-4-carboxylic acid. Dissolve this material in dilute hydrochloric acid at pH 2.5 and treat with charcoal (Darko KB) 8 for 5 minutes and then adjust the filtrate to pH 4 with 10% sodium hydroxide. Evaporate the Water at 40 at 15 mm. to give about one gram of 7-(D-a-amino-a-phenylacetamido) 3 (5 methyl-1,3,4-oxadiazol-2-ylthiomethyl)-3-cephcm-4-carboxylic acid.

EXAMPLE 2 Trichloroethyl 7 (D-2,2-dimethyl-3-nitroso 5 oxo-4- phenyl 1 imidazolidinyl)-3-rnethyl-3-cephem-4-carboxylate l-oxide To a solution of 1 g. (0.0024 mole) of 7-(D-2,2-di methyl 3 nitroso 5 ox0-4-phenyl-1-imidazolidinyl-3- methyl-3-cephem-4-carboxylic acid l-oxide dissolved 50 ml. of tetrahydrofuran was added 186 mg. (0.0024 mole) of pyridine, 750 mg. (0.0051 mole) of trichloroethanol and 500 mg. (0.0024 mole) of dicyclohexylcarbodiimide. The reaction mixture was stirred for 24 hours, filtered to remove the dicyclohexylurea and the filtrate was evaporated to a light yellow solid. This material was chromatographed over silic acid -200 mesh using methylene chloride (10)-acetone (I) solvent system. The initial fraction (30 ml.) was evaporated and the ester weighed 675 mg.; M.P. decomp.

Analysis.Calcd. for C H Cl N O S (percent): C, 44.73; H, 3.76; N, 9.94; Cl, 18.86. Found (percent): C, 44.99; H, 3.96; N, 9.93; Cl, 18.78.

NMR 100 mHz. (DMSO-d 7.5-7.1 (m, 5 HS, C 11 5.97 (d, J=4.5 Hz., 1 H, 0-7-5), 5.70 (S, 1 H, C H CE), 5.16 and 4.99 (AB doublets, J =12 Hz., 2 Hs, CO cg Ccl 4.90 (d, J=4.5 Hz., 0 11), 4.12 and 3.68 (AB doublets, J=16 Hz, 2 Hs, Cg 2.14, 2.03 and 2.08 (3, s, 9 Hs, C(Cg y C -CE IR (Nujol) 1800 cmr (e-lactam 0:0), 1730 (ester C=0, imidazolidinyl 0:0), 1065 (sulfoxide).

Trichloroethyl 7 (D-2,2-dimethyl 3 nitroso-5-oxo-4- phenyl-l-imidazolidinyl) 3 bromomethyl-3-cephem- 4-carboxylate l-oxide 0gb /H (I) 0 t-i t ON-N N l 7 CH3 CH3 0 N GHiBI Or-CHgCCh A 200 mg. (0.00356 mole) portion of the above ester was dissolved in 25 ml. of chloroform (treated with alumina to remove any methanol) and stirred at 20 with 71 mg. (0.004 mole) of N-bromo-succinimide and 2 mg. of benzoyl peroxide. The solution was illuminated for 4 hours under a photoflood lamp at 20. The chloroform was removed by distillation and the residue was dissolved in ethyl acetate and washed with Water. The organic phase was separated and evaporated to a gum which was dried by azeotropic distillation with ethyl acetate. The residue was dissolved in a minimal amount of ethyl acetate and diluted with Skellysolve B" to yield 188 mg.; M.P. 124 decomp.

Analysis.Calcd. for C H BrCl N O S (percent): C, 39.29; H, 3.14; N, 8.73. Found (percent): C, 39.87; H, 3.99; N, 8.42. Halogen equivalents 3.45.

This product is used to replace the free acid in the procedure of Example 1(b) and 1(c) to produce 7-(D-uamino-m-phenylacetamido)-3-(5 methyl 1,3,4 oxadiazol-Z-ylthiomethyl)-3-cephem-4-carboxylic acid.

9 EXAMPLE 3 (a) 7-(D-2,2-dimethyl 3 nitroso oxo-4-phenyl-1- imidazolidinyl) 3 methyl 3 cephem-4-carboxylic acid (N-nitrosohetacephalexin) from cephalexin To a mixture of g. (0.03 mole) of (7-D-u-aminophenylacetamido) 3 methyl 3 cephem-4-carboxylic acid (cephalexin) in 100 ml. of water was added 10% sodium hydroxide solution until a pH of 8.8 was attained. To this solution was added 40 ml. of acetone and the reaction was stored overnight. The solvent was evaporated leaving behind hetacephalexin as a frothy amorphous solid which was dissolved in 200 ml. of water and acidified to pH 2 with 6 N hydrochloric acid and layered with 200 ml. of ethyl acetate. The solution was cooled in an ice-bath to 5 and 1.6 g. (0.024 mole) of sodium nitrite was added. After stirring for /2 hour the ethyl acetate was separated, washed with water and evaporated under reduced pressure to an oil. The oil solidified on slurrying with ether to give 2.5 g. of N-nitrosohetacephalexin as an amorphous solid. During storage overnight a second crop separated which was crystalline and weighed 1.2 g. The crops were combined and recrystallized from ethyl acetate and ether. Yield 3.2 g. The analytical sample was recrystallized from boiling methanol, M.P. 8- 160.

Analysis.-Calcd. for C H N O S /2 H O (percent): C, 53.73; H, 4.74; N, 13.17. Found (percent): C, 53.90; H, 4.96; N, 13.48.

IR (KBr) 2500 to 3500 cm.- (carboxyl-OH), 1780 (B-lactam-FJ-N) 1720 and 1730 O O (imidazolidinoneN and carboxyl 700 (C H nMr (DMSO d 7.31 p.p.m. (S, 5,

5.55 (d, 1, J=4.5 c.p.s., NCECO), 5.15 (d, 1, 1:45 c.p.s., NCHS), 2.9 to 3.6

(In, 2, SCH: (m, 9, CfiaCEaCN and C111 /N CH3 CH3 CH8 To a solution of 500 mg. (0.013 mole) of 7-(D-2,2-dimethyl 3 nitroso 5 oxo-4-phenyl-l-imidazolidinyl)- 3-methyl-3-cephem-4-carboxylic acid in 20 ml. of water at pH 6 (adjusted by adding 1% NaOH solution) was added 300 mg. (0.014 mole) of sodium metaperiodate and the solution was stirred overnight. The mixture was filtered and the filtrate was acidified to pH 2 with a few drops of conc. hydrochloric acid. The solid was collected and recrystallized from acetone-water to yield 420 mg. (M.P. 188 decomp.) of crystalline 7-(D-2,2-dimethyl-3- nitroso 5 oxo 4'- phenyl-l-imidazolidinyl)-3-methyl- 3-cephem-4-carboxylic acid l-oxide.

Analysis.-Calcd. for C19H20N4O6S'CH3COCH3 (percent): C, 53.87; H, 5.34; N, 11.42. Found (percent): C, 53.61; H, 5.37; N, 11.42.

azolidinyl C=O), 1695 (acetone C=O), 1030 (sulfoxide), 700 (phenyl).

(c) 7-(D-2,2-dirnethyl-3-nitroso-5-oxo-4-phenyl-l-irnidazolidinyl)-3-bromomethyl-3-cephem-4-carboxylic acid 1- oxide To a solution of 900 mg. (.0022 mole) of the sulfoxide in 50 ml. of methylene chloride was added 218 mg. (.0022 mole) of triethylamine and 236 mg. (.0022 mole) of trimethylsilyl chloride. The mixture was stirred for /2 hour and cooled to 10 in an ice-bath. A total of 388 mg. (.0022 mole) of N-bromosuccinimide was added and the mixture was irradiated with a photoflood lamp for 2 hours. The methylene chloride was evaporated and the residue was slurried with water and collected to yield 425 mg. of 7 (D 2,2 dirnethyl-3-nitroso-5-oxo-4-phenyl-l-imidazolidinyl) 3 bromomethyl 3 cephem-4-carboxylic acid l-oxide; M.P. decomp.

Analysis.-Calcd. for C H BrN O S (percent): Br, 15.60. Found (percent): Br, 13.29.

IR (Nujol) 3100-3600 cm.- (carboxyl OH), 1785 (,8- lactam C=O), 1720 (carboxyl C=O, imidazolidinyl C=O), 1040 (sulfoxide).

This product is converted to 7-(D-2,2-dimethy1-3-nitroso 5 oxo 4 phenyl-1-imidazolidinyl)-3-(5-rneth yl-1,3,4-oxadiaz0l 2 ylthiomethyl)-3-cephem-4-carboxylic acid by silylation followed by reaction with potassium S-methyl-1,3,4-oxadiazole-Z-mercaptide according to the appropriate portions of Example 1(a).

EXAMPLE 4 7 (D 2,2 dimethyl-5-oxo-4-phenyl-l-imidazolidinyl)- 3 (5 methyl 1,3,4 oxadiazolyl-Z-thiomethyl)-3- cephem-4-carboxylic acid A solution of 200 mg. (0.005 mole) of 7-(D-2,2-dimethyl 3 nitroso 5 oxo-4-phenyl-1-imidazolidinyl)-3-(5- methyl 1,3,4 oxadiazolyl-Z-thiomethyl)-3-cephem-4- carboxylic acid in 25 ml. of dioxane (peroxide free) was treated with dry hydrogen chloride at 5 for 20 minutes. The solvent was evaporated under reduced pressure (15 mm. 25) and the residue was slurried with ethyl acetate and collected to yield 160 mg. 7-(D-2,2-dimethyl-5-oxo- 4 phenyl 1 imidazolidinyl) 3 (5-methyl-1,3,4-oxadiazolyl 2 thiomethyl)-3-cephem-4-carboxylic acid, M.P. The IR and NMR spectra were identical with the spectra of authentic material. A paper chromatogram of this material against a B. subtilus seeded agar plate developed in an n-butanol, ethanol, water system (4:1:5) showed a zone of inhibition corresponding exactly in Rf with that of an authentic specimen.

ALTERNATE PREPARATION OF COMPOUNDS USED FOR COMPARISON in 450 ml. of 0.1 M phosphate buffer, pH 6.4 followed by 15 g. (0.18 mole) of sodium bicarbonate. Then 9.5 g.

1 1 (0.09 mole) of 2-mercapto-5-methyl-1,3,4-oxadiazo1e was added and, with moderate stirring, the solution was heated to 60:0.5 C. At 60 C. the pH was checked and addi- 12 (c) 7-(D-2,2-dirnethyl 3 nitroso 5 oxo-4-pheny1-1- imidazolidinyl)-3-(5-methyl-1,3,4-oxadiazo1 2 ylthio methyl)-3-cephem-4-carboxylic acid tional sodium bicarbonate was added to readjust the pH to CiHs H 6.4. The solution was heated with stirring at 60:0.5 c. 5 11 for four hours. Then 18 g. of Darko KB" was added and s stirring was continued for 15 minutes. The solution was ON-N N f N N then filtered hot and a clear solution was obtained. The pH N I g g was adjusted to 4.5 by addition of 3 N HCl. After cooling CH CH CH S a a a at 0 C. in an ice bath for one hour the product was fil- 14] Y tered ofi" and washed with cold water. After air drying 5.1 g. was obtained, y c a solution of 1.0 g. (0.0021 mole) of 7-(D-a-am1no- A,wlysis cakdl for CHHI2N4O4S2 (percent): C a-phenylacetamido) 3 (5 methyl 1,3,4 oxadiazol-2- 40.23; H, 3.70; N, 17.06. Found (percent); C, 39.60; H ylthiomethyl)-3-cephem-4-carboxylic acid in 50 ml. of 4.18; N, 1581 Corrected for 0.7% 15 water adjusted to pH 9 by adding 10% NaOH dropwise The IR and NMR were consistent for the desired with stirring was added ml. of acetone. The solution product was stored at 5 over the Weekend. The acetone was evaporated 011 under reduced pressure (15 mm.) and (b) m 3 (s'methyl' 200 mg. (0.024 mole) of sodium nitrite was added. The 3 2 Ymnomethyl) 3 cephem solution was cooled in an ice-bath at 10 and ml. of carboxyh'c and ethyl acetate was added. After acidification with 1:1 phos- T a i d suspension f 525 (Q4323 mole) f phoric acid to pH 2 the ethyl acetate layer was separated, Sodi D- ;-[l-carbomethoxypropen 2 l i washed with water and azeotroped to 7-(D-2,2-dimethyl-3- phenylacetate in 70 m1. of acetonitrile and 2 drops of nitroso'5'oxo-4'phellyl 1 y y N,N-dimethylbenzy1amine at 10 c. was added 3 g. 1,3,4 vxadiazol 2 y e y 3 p (0.027 mole) of ethyl chloroformate and stirring continued bOXylic acid as a Yellow Solid which cfystalllled after for 15 minutes at 10 C. Next, a solution of 7.54 g. y 'g W Ethel Yield 471 2-; p- 0.023 mole) of 7-amino-3-(S-methyl-1,3,4oxadiazol-2- Analyw-Calcfor C22H22N6O6S (p r ylthiomethyl)-3-cephern-4-carboxylic acid in ml. of N, Found (P 4 acetonitrile, 30 ml. H 0 and 3.4 ml. (0.024 mole) of tri- 30 i Y ethylamine was precooled to 0 C. and added all at once IR l 3100-3600 y (B- and stirring continued for 30 minutes at 0 C. Salt (NaCl) l f (3:0), 1740 (carboxyl 1715 (lmldazol' was added in excess to saturate the solution (15 minutes) ldlnyl 0:0), 700 (P 3 and the organic layer was separated and to it added 25 (d) 1 3 i 5 4. h y1-1;. ml. of H O. The resulting solution was concentrated n imidazolidinyl) 3 (5 methy1 1,3,4 pzwmmvacuo at 22 C. to a volume of about 35 ml. To this methyl) 3 cephem 4 carboxylic acid 1 id aqueous solution was added a solution of 9 ml. of 90% CGHE H O formic acid in 75 ml. of M-IBK (methyl isobutyl ketone) U Q and the mixture was stirred for 30 minutes. Four grams I g of solids were filtered off and air dried. This material was 0NN N slurried in 35 ml. of H 0 and 5 m1. of H PO for N H 15 minutes, filtered and the filtrate stirred another 15 =-N HIP H: minutes with 2 g. of Darko KB carbon filtered again and A: finally the pH adjusted to 3.2 with NaI-ICO A small 2 amount of crystalline material was filtered off and dis- To a slurry of 4.5 g. (.0085 mole) of the above 7-(D- carded. The filtrate was concentrated slightly at reduced 2,2 dimethyl-3-nitroso-5-oxo-4-phenyl-1-imidazolidinyl)- pressure (22 C.) and a gummy solid precipitated. The 3-(5-methyl 1,3,4 oxadiazol 2 ylthiomethyl) 3- mixture was heated to C. whereupon the material cephem-4-carboxylic acid in 200 m1. of water was added (solids) crystallized rapidly. After slowly cooling to room 1.95 g. (.0093 mole) of sodium metaperiodate. The solutemperature there was obtained 1.24 g. of crystalline, white 50 tion was kept at pH 4.5 by the dropwise addition of 10% material, dec. pt. 165 C. NaOH for 4 hours. The mixture was filtered and the 7-[D (a-amino-a-phenylacetamido)]-3-(5-methyl 1,3, filtrate was acidified With 1:1 phosphoric acid to pH 2. 4 oxadiazol-2-ylthiomethyl)-3-cephem-4-carboxylic acid The crystalline solid was collected, washed well with water (called New Compound) after solution in 5% NaHCO and air dried to yield 3 g.; M.P. 207 decomp. The followed by dilution with Nutrient Broth was found to ex- 50 analytical sample was washed with methanol and dried in hibit the following Minimum Inhibitory Concentrations vacuo over P 0 for 24 hours. (M.I.C.) in mcg./ ml. versus the indicated microorganisms Analysis.Calcd. for C H N O S (percent): C, as determined by overnight incubation at 37 C. by Tube 48.34; H, 4.06; N, 15.38. Found (percent): C, 48.76; H, Dilution. Results with four old compounds are also given. I 4.19; N, 15.25.

TABLE 1 [M10 in meg/1111.]

New eorn- Cepha- Cephalo- Cepha- Cephalo- Organism pound lexin glycin lothin ridine D. pneumoniae plus 5% serum.. A9585 0. 13 1. 3 0. 3 0. 08 0. 008 Str. pyagenes plus 5% serum A9604 0.25 0. 3 0. 16 0. 08 0. 008 s. aureus Smith A9537 1.3 1.3 1.3 0. 0s 0. 03 S. aureus Smith plus 50% serum A9587 5 2. 5 2.5 0.3 0. 03 s. mucus BX1633-2 at 10- diln A9606 2. 5 4 0.6 0. a 0. a a S. aureus meth., resistant; 5 32 2 1. 3 0. 6 Sal. enteritidis 0. 6 4 0. 3 0. 3 0. 6 F. coliJuhl 4 8 1 4 1 F. coli 8 16 4 16 1 K. pneumon 1 4 0. 6 1 1. 3 D0 8 s 1 s 2 Pr. mirabilis 2 4 0. G 1 2. 5 Fr. morgam'i. 16 16 250 250 P. aeruginosa 250 125 250 250 250 Ser. marcescens 250 125 250 I 50% nutrient broth plus 45% antibiotic assay broth.

13 14 IR (Kbr) 2400-3400 cm.- (carboxyl OH), 1810 6. The compound of claim 5 having the D configuration lactam C=O), 1730 (carboxyl C=O, imidazolidinyl at the carbon atom adjacent to the benzene ring. C=O), 1025 (sulfoxide), 705 (phenyl). 7. The compound of the formula We claim: 1. A compound having the formula 5 A, H O C} H G g 1 Orr-1 1 1 I ON-N N X 5'; Z on, ona o 3 0H, O=- --N CHz-Br (503E: OgCHzCCl; wherein Ar is phenyl, Z-thienyl or 3-thienyl.

A compound havmg the formula 8. Thecompound of claim 7 having the D configuration Ar H O at the carbon atom adjacent to the benzene ring.

g. 9. The compound of the formula ON-N 1 1 X i l C 3 CH3 Y-CHa-Br /H (A? 0 O I 0 011- 0015 ON Ig IlI wherein Ar 1s phenyl, 2-th1enyl or 3-th1enyl. I I I 3. A compound having the formula 0= N B CH on, Ar H O 1 5 2R ON--N N J 1 wherein R is a silyl group. o N 10. The compound of claim 9 having the formula CH3 CH3 2 /OzR wherein Ar is phenyl, Z-thienyl 0r 3-thienyl and R is a Q H 0 silyl group. g

4. d h l A comfoun H aging the formu a I Y I l I I 0 0L I cnf CH: ON-N N f l 2 mm): N

CHIBI' 11. The compound of claim 10 having the D configurao sucrm tion at the carbon atom adjacent to the benzene ring. wherein Ar is phenyl, Z-thienyl or S-thienyl.

5. The compound of the formula References cued UNITED STATES PATENTS Q H 0 3,634,418 1/1972 Willner 260-243 c a 0 3,647,786 3/1972 Cooper 260-243 C 3 3,655,658 4/1972 Godtfredsen et al. 260-243 c ON-N N X 0 N NICHOLAS S. RIZZO, Primary Examiner CH2BX CH3 CH3 US. Cl. X.R. 

